Mass spectrometry has become the method of choice for fast and efficient identification of proteins in biological samples. In general, a mass spectrometer comprises an ion source for generating ions from molecules to be analyzed, and ion optics for guiding the ions to a mass analyzer. A tandem mass spectrometer further comprises the ability to perform a second stage of mass analysis. As is well known, the second stage of mass analysis may be performed using a tandem-in-space configuration employing more than one analyzer or in a tandem-in-time configuration, using only a single analyzer. In tandem mass spectrometry, structural elucidation of ionized molecules is performed by collecting a mass spectrum, then using a first mass analyzer to select a desired precursor ion or ions from the mass spectrum, causing fragmentation of the selected precursor ions, and then performing mass analysis of the fragment ions using a second stage of mass analysis.
Tandem mass spectrometry of peptides in a complex protein mixture can be used to identify and quantify the proteins present in the original mixture. Tandem mass spectrometers achieve this by selecting single m/z values and subjecting the precursor ions to fragmentation, providing product ions that can be used to sequence and identify peptides. This method can be extended to provide one or more further stages of fragmentation (i.e. fragmentation of fragment ions and so on). This is typically referred to as MSn, with the superscript “n” denoting the number of generations of ions. Thus MS2 corresponds to tandem mass spectrometry.
In many research and clinical applications, it is desirable to not only to identify peptides or proteins according to their amino acid composition, but also to quantify these analytes according to their respective sources. Isotopic labels have been extensively employed for the latter purpose. Recently, labels which are categorized as so-called “isobaric tags” have been developed in order to overcome the problems that: (a) native peptides and deuterium-labeled peptides frequently do not precisely co-elute (b) the isotopically-labeled peptides often give rise to charge states, upon electrospray ionization, that are different from those of the non-labeled peptides (A. Thompson et al., “Tandem Mass Tags: A Novel Quantification Strategy for Comparative Analysis of Complex Protein Mixtures by MS/MS”, Anal. Chem. 2003, 75, 1895-1904).
A first isobaric labeling technique employs sets of reagents known as tandem mass tags (ibid.) which provide labels comprising a mass reporter region, a cleavable linker region, a mass-normalization region and a reactive group. Different samples of a single peptide, when labeled with different respective tandem mass tag (TMT) reagents, will all comprise the same chemical structure and mass and will therefore co-elute. However, upon mass spectrometric analysis by collision-induced dissociation, an ion is released that has a specific mass-to-charge ratio that is diagnostic of a specific tag, thereby enabling identification of the source of the peptide or protein. A second isobaric labeling technique, known as “isobaric tags for relative and absolute quantitation” or (iTRAQ) employs isobaric mass labels attached to the N termini and lysine side chains of peptides in a digest mixture. As in the TMT technique, all differentially-labeled samples of a single analyte will comprise the same chemical structure and mass. Upon undergoing collision induced dissociation, however, signature or reporter ions are released that can be used to identify and quantify individual proteins according to their respective sources or treatments. The iTRAQ technique is described in P. L. Ross et al., “Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents”, Molecular and Cellular Proteomics 2004, 3, 1154-1169.
In either the TMT or iTRAQ technique, precursor ions comprising particular mass-to-charge (m/z) ratios corresponding to expected or known peptides are isolated. These precursor ions are then fragmented by collision-induced dissociation so as to both cleave the peptide backbone as well to fragment the tags. Sequence information may then be obtained from analysis of the fragments produced by the backbone cleavage and source identification may be revealed by analysis of reporter ions produced by fragmentation of the tags.
Recently, it has been realized that the use of isobaric tags for quantitation of peptides and proteins is affected by inherent interferences that fall within the initial m/z isolation window. These interferences are due to unrelated co-eluting peptides that fall within the isolation window for the precursor of interest. Since the vast majority of peptides have unchanged ratios between control and experiment, the interferences tend to drag all ratios towards unity, and thus its difficult to obtain accurate quantitative values.
Several approaches have been proposed to minimize the impact of the interference. A first approach (G. M. Sweetman, “Synchronising MS/MS Analysis with The Chromatographic Peak Apex Enables More Precise and Accurate Isobaric Tag Quantification”, Proc. 58th ASMS, Salt Lake City, 2010) employs triggering mass analysis at the apex of targeted chromatographic peaks (LC apex triggering), which in theory enhances the purity of the precursor, since it is examined when it is most concentrated. A second proposed approach includes the use of proton transfer reagents to reduce ion charge state and shift t precursor m/z, followed by an additional step of isolation before fragmentation (D. Bailey et al., “How High Mass Accuracy Measurements Will Transform Targeted Proteomics”, 8th North American FT MS Conference, Key West, Fla., 2011), In another approach, termed “Quantmode”, isolation purity filters are employed (C. D. Wenger et al., “A real-time data acquisition method for improved protein quantitation on hybrid mass spectrometers”, Proc. 58th ASMS, Salt Lake City, 2010). The Quantmode technique involves triggering MS/MS analysis only for precursors that meet a defined purity within the intended isolation window. All the above methods provide some level of reduced interference, but still show systematic deviation of ratios towards unity.
In yet another approach (L. Ting et al., “MS 3 Eliminates Ratio Distortion in Isobaric Multiplexed Quantitative Proteomics”, Nature Methods 2011, 8, 937-940) the use of MS3 is reported. In this technique, a peptide ion is first isolated and fragmented. A selected fragment of the precursor is then itself isolated, and higher-energy dissociation (HCD) of the isolated fragment is performed to release the tag. This method is only successful because most fragments formed by ion trap CID maintain the tag. Although this method provides the most accurate tag ratios to date, it suffers from poor sensitivity, since any particular fragment is rarely more than 5% of the abundance of the initial precursor. Accordingly, there remains a need in the art for a method of analyzing isobarically tagged proteins and peptides that both minimizes interference and provides high sensitivity.